Large-scale DNA sequencing in diagnostic and prognostic applications has expanded rapidly as its speed and convenience has increased and its per-base cost has decreased, e.g. Ding et al, Nature, 481(7382): 506-510 (2012); Chiu et al, Brit. Med. J., 342: c7401 (2011); Ku et al, Annals of Neurology, 71(1): 5-14 (2012); and the like. In particular, profiles of nucleic acids encoding immune molecules, such as T cell or B cell receptors, or their components, contain a wealth of information on the state of health or disease of an organism, so that the use of such profiles as diagnostic or prognostic indicators has been proposed for a wide variety of conditions, e.g. Faham and Willis, U.S. Pat. Nos. 8,236,503 and 8,628,927; Freeman et al, Genome Research, 19: 1817-1824 (2009); Han et al, J. Immunol, 182 (1001): 42.6 (2009); Boyd et al, Sci. Transl. Med., 1(12): 12ra23 (2009); He et al, Oncotarget (Mar. 8, 2011).
For example, patients treated for many cancers often retain a minimal residual disease (MRD) related to the cancer. That is even though a patient may have by clinical measures a complete remission of the disease in response to treatment, a small fraction of the cancer cells may remain that have, for one reason or another, escaped destruction. The type and site of this residual population is an important prognostic factor for the patient's continued treatment, e.g. Campana. Hematol. Oncol. Clin. North Am., 23(5): 1083-1098 (2009); Buccisano et al, Blood, 119(2): 332-341 (2012). Consequently, several techniques for assessing this population have been developed, including techniques based on flow cytometry, in situ hybridization, cytogenerics, amplification of nucleic acid markers, and the like, e.g. Buccisano et al, Current Opinion in Oncology, 21: 582-588 (2009); van Dongen et al, Leukemia, 17(12): 2257-2317 (2003); and the like. The amplification of recombined nucleic acids encoding segments of immune receptors (i.e. clonotypes) from T cells and/or B cells have been particularly useful in assessing MRD in leukemias and lymphomas, because such clonotypes typically have unique sequences which may serve as molecular tags for their associated cancer cells. Such measurements are usually made by amplifying and sequencing, nucleic acids encoding a single receptor chain, in part, because such amplifications are highly multiplexed and are difficult to develop. As the scale of multiplexing increases, several problems are encountered, including increased probability of spurious amplifications due to mis-hybridizations, primer-dimer formation, variable rates of amplification leading to biased sequence representation, and the like, e.g. Elnifro et al, Clinical Microbiology Reviews, 13(4): 559-570 (2000). Furthermore, the similarity of the target sequences and the incorporation of sequence tags into amplified sequences, either for sequence analysis, sample tracking, contamination detection, or the like, can exacerbate the above difficulties associated with large-scale amplifications. These challenges have prevented the development of large-scale one-reaction amplifications of multiple immune receptor chains, which would be highly beneficial for reducing the number of separate assays required for measuring nucleic acid sequences correlated with a minimal disease.
In view of the foregoing, it would be highly advantageous if more efficient methods were available for assessing selected nucleic acids in a single reaction, such as exons of cancer genes or clonotypes encoding sets of immune receptor chains.